G Model
PHAREP 67 1–8 Pharmacological Reports xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep
Original research article
Opioid modulation of prolactin secretion induced by stress during late pregnancy. Role of ovarian steroids Susana R. Valdez a,c,1, Gisela E. Pennacchio a,1, Dante F. Gamboa a, Elina G. de Di Nasso a, Claudia Bregonzio b, Marta Soaje a,d,* a
Laboratorio de Reproduccio´n y Lactancia, IMBECU-CONICET, Mendoza, Argentina Departamento de Farmacologı´a, Facultad de Ciencias Quı´micas, Universidad Nacional de Co´rdoba, Argentina Instituto de Ciencias Ba´sicas, Universidad Nacional de Cuyo, Mendoza, Argentina d Departamento de Morfofisiologı´a, Facultad de Ciencias Me´dicas, Universidad Nacional de Cuyo, Mendoza, Argentina b c
A R T I C L E I N F O
Article history: Received 28 March 2013 Received in revised form 6 December 2013 Accepted 20 December 2013 Available online xxx Keywords: Prolactin Stress Naloxone Pregnancy Opioid receptor subtypes
A B S T R A C T
Background: The opioid system modulates prolactin release during late pregnancy. Its role and the participation of ovarian hormones in this modulation are explored in ether stress-induced prolactin release. Methods/Results: Estrous, 3-day and 19-day pregnant rats were used. We administered the antagonist mifepristone (Mp) and tamoxifen to evaluate progesterone and estradiol action in naloxone (NAL, opioid antagonist) or saline treated rats. Ether stress had no effect on serum prolactin levels in controls but increased prolactin release in NAL-treated rats. Prolactin response to stress in NAL-treated rats was blocked by L-DOPA administration. Mp treatment on day 18 of pregnancy increased prolactin levels after stress without alterations by NAL. Tamoxifen on days 14 and 15 of pregnancy completely blocked Mp and NAL effects on prolactin release at late pregnancy. In contrast, stress significantly increased prolactin levels in estrous rats and pretreatment with NAL prevented this. On day 3 of pregnancy, at 6.00 p.m., stress and NAL treatment inhibited prolactin levels in saline-treated rat. No effect of stress or NAL administration was detected on day 3 of pregnancy at 9.00 a.m. icv administration of specific opioids antagonist, B-Funaltrexamine but not Nor-Binaltorphimine or Naltrindole, caused a significant increase in stress-induced prolactin release. Conclusions: Opioid system suppression of prolactin stress response during late pregnancy was observed only after progesterone withdrawal, involving a different opioid mechanism from its well-established stimulatory role. This mechanism acts through a mu opioid receptor and requires estrogen participation. The opioid system and progesterone may modulate stress-induced prolactin release, probably involving a putative prolactin-releasing factor. ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Introduction The opioid system modulates prolactin secretion in nonpregnant, pregnant and lactating animals [1–6]. Dual neuromodulatory regulation of prolactin secretion by the opioid system during pregnancy was previously described [7]. After stimulatory action during the first days, a change to inhibitory control was established at the end of pregnancy, starting around day 16 [8].
* Corresponding author. E-mail address: [email protected] (M. Soaje). 1 S.R. Valdez and G.E. Pennacchio contributed equally to this work.
Thus, both stimulatory and inhibitory actions of opioids, acting through different regulatory pathways, may result in an elevation of prolactin levels [7,9,10]. Administration of the antiprogesterone mifepristone (Mp) facilitates prolactin release by blocking the central inhibitory action of progesterone [7], and the effect of Mp can be enhanced by injecting the opioid antagonist naloxone (NAL). In fact, prolactin secretion during late pregnancy undergoes a paradoxical regulation by the opioid system in which progesterone plays an important role [7,8]. Moreover, Mp inhibits the hypothalamic dopaminergic neuronal system [11], the main inhibitory factor of prolactin secretion in terms of dopaminergic transmission and tyrosine hydroxylase (TH) expression [12,13]. This effect enables a
http://dx.doi.org/10.1016/j.pharep.2013.12.006 1734-1140/ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Please cite this article in press as: Valdez SR, et al. Opioid modulation of prolactin secretion induced by stress during late pregnancy. Role of ovarian steroids. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2013.12.006
G Model
PHAREP 67 1–8 2
S.R. Valdez et al. / Pharmacological Reports xxx (2014) xxx–xxx
significant activation of lactotrophs and primes the pituitary for a subsequent stimulatory action of NAL [14]. Several stressors may affect prolactin secretion [15–17], and endogenous opioid peptides participate in the prolactin response to stress [18–20]. Among the subtypes of opioid receptors (mu, delta and kappa receptors), the activation of mu opioid subtypes plays an important role in stress conditions [21,22]. Furthermore, ether stress induces a rapid increase in plasma prolactin concentrations in female, male, and androgenized rats [23], and ovarian steroids participate in this effect [24,25]. It is known that estradiol has a stimulatory effect on basal as well as on stressinduced prolactin release [26,27] and evidence suggests that progesterone may inhibit prolactin gene expression [28] and prolactin secretion [24] in response to stress. The mechanisms by which endogenous opioids and ovarian steroids may affect stress responsiveness are, however, still unclear. Interestingly, hyporesponsiveness of the hypothalamus–pituitary–adrenal (HPA) axis to several stressors was described in late pregnancy [29]. Both endogenous opioids and progesterone, more specifically its metabolite allopregnanolone [30], seem to play an important role in the mechanisms involved in this suppressed HPA axis response [24,31]. Several changes occur in the maternal brain to prepare the different neuroendocrine systems involved in mechanisms regulating parturition and lactation [32,33]. Among others, endogenous opioids and progesterone play a role in maternal oxytocin and prolactin system adaptation [31,32]. The primary goal of this study was to examine the participation of the opioid system in the regulation of prolactin secretion in response to ether stress during late pregnancy and to establish a correlation with changes in ovarian steroids. Additionally, the ether stress response in other reproductive situations was evaluated, such as estrus day or day 3 of pregnancy where the stimulatory effect of the opioid system has been clearly established. Materials and methods Animals Virgin female rats, 3 months old (200–220 g), bred in our laboratory and originally from the Wistar strain, were used. They were kept in a light (6.00 a.m. – 8.00 p.m.) and temperature (22 2 8C)-controlled room. Rat chow (Cargill, Argentina) and tap water were available ad libitum. Vaginal smears were analyzed daily; virgin rats of 3 months of age showing two or three consecutive 4-day cycles were used on estrus day. Other groups of rats were made pregnant by being caged individually with a fertile male on the night of proestrus. Vaginal smears were checked for the presence of spermatozoa on the following morning; if positive, that day was considered day 0 of pregnancy (normal delivery on day 22 of pregnancy). Animal maintenance and handling were conducted according to the NIH guide for the Care and Use of Laboratory Animals (NIH publication N8 86-23, revised 1985 and 1991) and the UK requirements for ethics of animal experimentation (Animals Scientific Procedures, Act 1986). All experimental procedures were approved by the Care and Use of Laboratory Animals Committee (CICUAL) of the Faculty of Medical Sciences, National University of Cuyo, Mendoza, Argentina. Surgical procedures In pregnant rats receiving intracerebroventricular (icv) injections, stainless-steel guide cannulae were surgically implanted on day 12, 7 days before the experiment. The animals were anesthetized with a combination of xylazine hydrochloride (4 mg/kg) and ketamine hydrochloride (80 mg/kg) injected ip between 9.00 a.m. and 12.00 a.m. Rats were positioned in a
stereotaxic frame and the stainless-steel guide cannula was inserted into the right lateral ventricle (M/L 1.5 mm, A/P0.4 mm relative to bregma, 4.5 mm relative to dura [34]). Cannulae were fixed to the skull using dental acrylic and sealed until the time of drug injection. On the day of the experiment, 5 ml of specific antagonists were injected using a 10 ml Hamilton microsyringe connected to an injection needle that protruded 1 mm beyond the tip of the guide cannula placed in the lateral ventricle. Placement of cannulae was verified histologically at the end of the experiment. Drugs The opioid receptor antagonists m: Beta-Funaltrexamine (BFNA), k: Nor-Binaltorphimine (Nor-BNI), d: Naltrindole (NALT) and the non-specific opioid receptor antagonist: Naloxone (NAL), and the antiprogesterone mifepristone (Mp) (RU-486:17b-hydroxy11b-[4-dimethyl-amino-phenyl]-17a-propinyl-estra-4,9-dien-3one); were obtained from Sigma Chemical Co, St Louis, MO, USA. Tamoxifen citrate (T) was provided by Gador S.A., Buenos Aires, Argentina. L-dihydroxyphenylalanine (L-DOPA) was obtained from Roche, Buenos Aires, Argentina. Exposure to stress Rats were placed individually in a jar saturated with ether vapor for 2 min [24,35]. Blood samples were obtained by decapitation 3 min after ether exposure. The experiments were conducted at 9.00 a.m. except on day 3 of pregnancy when they were also conducted at 6.00 p.m. (surge prolactin time). Control rats were always included. Several studies suggest that maximum prolactin response is reached between 2 and 5 min after ether exposure [24,25,35,36]. The order of decapitation did not affect circulating hormone levels of the basal or stressed rats groups. Blood samples were collected without anticoagulant and allowed to clot at room temperature. Serum was separated by centrifugation and stored frozen at 20 8C until subsequent radioimmunoassay. Experimental procedures Experiment 1 This experiment was designed to establish the effect of ether stress on prolactin secretion in NAL-treated rats at late pregnancy. We also included, in the present experiment, two other groups of rats on estrous day and day 3 of pregnancy where the stimulatory effect of the opioid system has been clearly established. Animals on day 19 of pregnancy (late pregnancy), day 3 of pregnancy or estrus day received an ip injection of NAL (2 mg/kg) or its vehicle (SAL) at 8.30 a.m. and were sacrificed at 9.00 a.m. Five minutes prior to decapitation, the rats were exposed to ether vapors as described above. Another group of rats was sacrificed following the same schedule at 6.00 p.m. on day 3 of pregnancy when serum prolactin levels are elevated. It is known that in most situations, prolactin release is under an inhibitory dopaminergic tone of hypothalamic origin. Rats on day 19 of pregnancy were treated with saline or the dopamine precursor LDOPA (25 mg/kg, ip) at 8.15 a.m. to prevent any transient decrease of dopaminergic tone, 15 min later with NAL or saline, and following the same stress exposure they were sacrificed at 9.00 a.m. Blood samples were obtained to determine serum prolactin and progesterone concentrations by radioimmunoassay (RIA). Experiment 2 This experiment was conducted to study the effect of the fluctuations in progesterone and estrogen action occurring during
Please cite this article in press as: Valdez SR, et al. Opioid modulation of prolactin secretion induced by stress during late pregnancy. Role of ovarian steroids. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2013.12.006
G Model
PHAREP 67 1–8 S.R. Valdez et al. / Pharmacological Reports xxx (2014) xxx–xxx
3
late pregnancy on the modulation of endogenous opioids on stressinduced prolactin secretion. Tamoxifen citrate (T, antagonist of estrogen receptor) 500 mg/ kg dissolved in 0.14 M NaCl, 0.5% (v/v) Tween 80 or vehicle was orally administered at 4.00 p.m. on days 14 and 15 of pregnancy, when estrogen levels begin to increase until reaching their maximum prior to delivery [37]. On day 18 of pregnancy, rats were treated with mifepristone (Mp, 2 mg/kg, sc) or vehicle (oil) at 9.00 p.m. On day 19 of pregnancy, all rats received an ip injection of NAL (2 mg/kg) or vehicle (SAL) at 8.30 a.m., and were sacrificed at 9.00 a.m. Five minutes prior to decapitation, they were exposed to ether vapors as above described. Blood samples were obtained for serum prolactin determination by RIA. Experiment 3 This experiment was conducted to identify opioid receptor subtypes involved in prolactin release regulation in rats exposed to ether stress in late pregnancy. The irreversible mu opioid receptor antagonist B-FNA (5 mg/rat), kappa antagonist Nor-BNI (15 mg/ rat), and delta antagonist NALT (5 mg/rat) were dissolved in distilled water immediately before administration. Drugs or vehicle were administered intracerebroventricularly (icv) 30 min before sacrifice. Five minutes prior to decapitation, rats were exposed to ether vapors as described. Blood samples were obtained for serum prolactin and progesterone determination by RIA. Prolactin and progesterone determinations Serum prolactin concentration was measured by doubleantibody radioimmunoassay [8] with materials supplied by Dr. A. F. Parlow (National Hormone and Pituitary Program, USA). Prolactin was radioiodinated using the chloramine T method [38] and purified by passage through Sephadex G-75 and polyacrylamide agarose (ACA 54; LKB, Bromma, Sweden) columns. Assay sensitivity was 1 ng/ml serum, and inter- and intra-assay coefficients of variation were
PHAREP 67 1–8 Pharmacological Reports xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep
Original research article
Opioid modulation of prolactin secretion induced by stress during late pregnancy. Role of ovarian steroids Susana R. Valdez a,c,1, Gisela E. Pennacchio a,1, Dante F. Gamboa a, Elina G. de Di Nasso a, Claudia Bregonzio b, Marta Soaje a,d,* a
Laboratorio de Reproduccio´n y Lactancia, IMBECU-CONICET, Mendoza, Argentina Departamento de Farmacologı´a, Facultad de Ciencias Quı´micas, Universidad Nacional de Co´rdoba, Argentina Instituto de Ciencias Ba´sicas, Universidad Nacional de Cuyo, Mendoza, Argentina d Departamento de Morfofisiologı´a, Facultad de Ciencias Me´dicas, Universidad Nacional de Cuyo, Mendoza, Argentina b c
A R T I C L E I N F O
Article history: Received 28 March 2013 Received in revised form 6 December 2013 Accepted 20 December 2013 Available online xxx Keywords: Prolactin Stress Naloxone Pregnancy Opioid receptor subtypes
A B S T R A C T
Background: The opioid system modulates prolactin release during late pregnancy. Its role and the participation of ovarian hormones in this modulation are explored in ether stress-induced prolactin release. Methods/Results: Estrous, 3-day and 19-day pregnant rats were used. We administered the antagonist mifepristone (Mp) and tamoxifen to evaluate progesterone and estradiol action in naloxone (NAL, opioid antagonist) or saline treated rats. Ether stress had no effect on serum prolactin levels in controls but increased prolactin release in NAL-treated rats. Prolactin response to stress in NAL-treated rats was blocked by L-DOPA administration. Mp treatment on day 18 of pregnancy increased prolactin levels after stress without alterations by NAL. Tamoxifen on days 14 and 15 of pregnancy completely blocked Mp and NAL effects on prolactin release at late pregnancy. In contrast, stress significantly increased prolactin levels in estrous rats and pretreatment with NAL prevented this. On day 3 of pregnancy, at 6.00 p.m., stress and NAL treatment inhibited prolactin levels in saline-treated rat. No effect of stress or NAL administration was detected on day 3 of pregnancy at 9.00 a.m. icv administration of specific opioids antagonist, B-Funaltrexamine but not Nor-Binaltorphimine or Naltrindole, caused a significant increase in stress-induced prolactin release. Conclusions: Opioid system suppression of prolactin stress response during late pregnancy was observed only after progesterone withdrawal, involving a different opioid mechanism from its well-established stimulatory role. This mechanism acts through a mu opioid receptor and requires estrogen participation. The opioid system and progesterone may modulate stress-induced prolactin release, probably involving a putative prolactin-releasing factor. ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Introduction The opioid system modulates prolactin secretion in nonpregnant, pregnant and lactating animals [1–6]. Dual neuromodulatory regulation of prolactin secretion by the opioid system during pregnancy was previously described [7]. After stimulatory action during the first days, a change to inhibitory control was established at the end of pregnancy, starting around day 16 [8].
* Corresponding author. E-mail address: [email protected] (M. Soaje). 1 S.R. Valdez and G.E. Pennacchio contributed equally to this work.
Thus, both stimulatory and inhibitory actions of opioids, acting through different regulatory pathways, may result in an elevation of prolactin levels [7,9,10]. Administration of the antiprogesterone mifepristone (Mp) facilitates prolactin release by blocking the central inhibitory action of progesterone [7], and the effect of Mp can be enhanced by injecting the opioid antagonist naloxone (NAL). In fact, prolactin secretion during late pregnancy undergoes a paradoxical regulation by the opioid system in which progesterone plays an important role [7,8]. Moreover, Mp inhibits the hypothalamic dopaminergic neuronal system [11], the main inhibitory factor of prolactin secretion in terms of dopaminergic transmission and tyrosine hydroxylase (TH) expression [12,13]. This effect enables a
http://dx.doi.org/10.1016/j.pharep.2013.12.006 1734-1140/ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Please cite this article in press as: Valdez SR, et al. Opioid modulation of prolactin secretion induced by stress during late pregnancy. Role of ovarian steroids. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2013.12.006
G Model
PHAREP 67 1–8 2
S.R. Valdez et al. / Pharmacological Reports xxx (2014) xxx–xxx
significant activation of lactotrophs and primes the pituitary for a subsequent stimulatory action of NAL [14]. Several stressors may affect prolactin secretion [15–17], and endogenous opioid peptides participate in the prolactin response to stress [18–20]. Among the subtypes of opioid receptors (mu, delta and kappa receptors), the activation of mu opioid subtypes plays an important role in stress conditions [21,22]. Furthermore, ether stress induces a rapid increase in plasma prolactin concentrations in female, male, and androgenized rats [23], and ovarian steroids participate in this effect [24,25]. It is known that estradiol has a stimulatory effect on basal as well as on stressinduced prolactin release [26,27] and evidence suggests that progesterone may inhibit prolactin gene expression [28] and prolactin secretion [24] in response to stress. The mechanisms by which endogenous opioids and ovarian steroids may affect stress responsiveness are, however, still unclear. Interestingly, hyporesponsiveness of the hypothalamus–pituitary–adrenal (HPA) axis to several stressors was described in late pregnancy [29]. Both endogenous opioids and progesterone, more specifically its metabolite allopregnanolone [30], seem to play an important role in the mechanisms involved in this suppressed HPA axis response [24,31]. Several changes occur in the maternal brain to prepare the different neuroendocrine systems involved in mechanisms regulating parturition and lactation [32,33]. Among others, endogenous opioids and progesterone play a role in maternal oxytocin and prolactin system adaptation [31,32]. The primary goal of this study was to examine the participation of the opioid system in the regulation of prolactin secretion in response to ether stress during late pregnancy and to establish a correlation with changes in ovarian steroids. Additionally, the ether stress response in other reproductive situations was evaluated, such as estrus day or day 3 of pregnancy where the stimulatory effect of the opioid system has been clearly established. Materials and methods Animals Virgin female rats, 3 months old (200–220 g), bred in our laboratory and originally from the Wistar strain, were used. They were kept in a light (6.00 a.m. – 8.00 p.m.) and temperature (22 2 8C)-controlled room. Rat chow (Cargill, Argentina) and tap water were available ad libitum. Vaginal smears were analyzed daily; virgin rats of 3 months of age showing two or three consecutive 4-day cycles were used on estrus day. Other groups of rats were made pregnant by being caged individually with a fertile male on the night of proestrus. Vaginal smears were checked for the presence of spermatozoa on the following morning; if positive, that day was considered day 0 of pregnancy (normal delivery on day 22 of pregnancy). Animal maintenance and handling were conducted according to the NIH guide for the Care and Use of Laboratory Animals (NIH publication N8 86-23, revised 1985 and 1991) and the UK requirements for ethics of animal experimentation (Animals Scientific Procedures, Act 1986). All experimental procedures were approved by the Care and Use of Laboratory Animals Committee (CICUAL) of the Faculty of Medical Sciences, National University of Cuyo, Mendoza, Argentina. Surgical procedures In pregnant rats receiving intracerebroventricular (icv) injections, stainless-steel guide cannulae were surgically implanted on day 12, 7 days before the experiment. The animals were anesthetized with a combination of xylazine hydrochloride (4 mg/kg) and ketamine hydrochloride (80 mg/kg) injected ip between 9.00 a.m. and 12.00 a.m. Rats were positioned in a
stereotaxic frame and the stainless-steel guide cannula was inserted into the right lateral ventricle (M/L 1.5 mm, A/P0.4 mm relative to bregma, 4.5 mm relative to dura [34]). Cannulae were fixed to the skull using dental acrylic and sealed until the time of drug injection. On the day of the experiment, 5 ml of specific antagonists were injected using a 10 ml Hamilton microsyringe connected to an injection needle that protruded 1 mm beyond the tip of the guide cannula placed in the lateral ventricle. Placement of cannulae was verified histologically at the end of the experiment. Drugs The opioid receptor antagonists m: Beta-Funaltrexamine (BFNA), k: Nor-Binaltorphimine (Nor-BNI), d: Naltrindole (NALT) and the non-specific opioid receptor antagonist: Naloxone (NAL), and the antiprogesterone mifepristone (Mp) (RU-486:17b-hydroxy11b-[4-dimethyl-amino-phenyl]-17a-propinyl-estra-4,9-dien-3one); were obtained from Sigma Chemical Co, St Louis, MO, USA. Tamoxifen citrate (T) was provided by Gador S.A., Buenos Aires, Argentina. L-dihydroxyphenylalanine (L-DOPA) was obtained from Roche, Buenos Aires, Argentina. Exposure to stress Rats were placed individually in a jar saturated with ether vapor for 2 min [24,35]. Blood samples were obtained by decapitation 3 min after ether exposure. The experiments were conducted at 9.00 a.m. except on day 3 of pregnancy when they were also conducted at 6.00 p.m. (surge prolactin time). Control rats were always included. Several studies suggest that maximum prolactin response is reached between 2 and 5 min after ether exposure [24,25,35,36]. The order of decapitation did not affect circulating hormone levels of the basal or stressed rats groups. Blood samples were collected without anticoagulant and allowed to clot at room temperature. Serum was separated by centrifugation and stored frozen at 20 8C until subsequent radioimmunoassay. Experimental procedures Experiment 1 This experiment was designed to establish the effect of ether stress on prolactin secretion in NAL-treated rats at late pregnancy. We also included, in the present experiment, two other groups of rats on estrous day and day 3 of pregnancy where the stimulatory effect of the opioid system has been clearly established. Animals on day 19 of pregnancy (late pregnancy), day 3 of pregnancy or estrus day received an ip injection of NAL (2 mg/kg) or its vehicle (SAL) at 8.30 a.m. and were sacrificed at 9.00 a.m. Five minutes prior to decapitation, the rats were exposed to ether vapors as described above. Another group of rats was sacrificed following the same schedule at 6.00 p.m. on day 3 of pregnancy when serum prolactin levels are elevated. It is known that in most situations, prolactin release is under an inhibitory dopaminergic tone of hypothalamic origin. Rats on day 19 of pregnancy were treated with saline or the dopamine precursor LDOPA (25 mg/kg, ip) at 8.15 a.m. to prevent any transient decrease of dopaminergic tone, 15 min later with NAL or saline, and following the same stress exposure they were sacrificed at 9.00 a.m. Blood samples were obtained to determine serum prolactin and progesterone concentrations by radioimmunoassay (RIA). Experiment 2 This experiment was conducted to study the effect of the fluctuations in progesterone and estrogen action occurring during
Please cite this article in press as: Valdez SR, et al. Opioid modulation of prolactin secretion induced by stress during late pregnancy. Role of ovarian steroids. Pharmacol Rep (2014), http://dx.doi.org/10.1016/j.pharep.2013.12.006
G Model
PHAREP 67 1–8 S.R. Valdez et al. / Pharmacological Reports xxx (2014) xxx–xxx
3
late pregnancy on the modulation of endogenous opioids on stressinduced prolactin secretion. Tamoxifen citrate (T, antagonist of estrogen receptor) 500 mg/ kg dissolved in 0.14 M NaCl, 0.5% (v/v) Tween 80 or vehicle was orally administered at 4.00 p.m. on days 14 and 15 of pregnancy, when estrogen levels begin to increase until reaching their maximum prior to delivery [37]. On day 18 of pregnancy, rats were treated with mifepristone (Mp, 2 mg/kg, sc) or vehicle (oil) at 9.00 p.m. On day 19 of pregnancy, all rats received an ip injection of NAL (2 mg/kg) or vehicle (SAL) at 8.30 a.m., and were sacrificed at 9.00 a.m. Five minutes prior to decapitation, they were exposed to ether vapors as above described. Blood samples were obtained for serum prolactin determination by RIA. Experiment 3 This experiment was conducted to identify opioid receptor subtypes involved in prolactin release regulation in rats exposed to ether stress in late pregnancy. The irreversible mu opioid receptor antagonist B-FNA (5 mg/rat), kappa antagonist Nor-BNI (15 mg/ rat), and delta antagonist NALT (5 mg/rat) were dissolved in distilled water immediately before administration. Drugs or vehicle were administered intracerebroventricularly (icv) 30 min before sacrifice. Five minutes prior to decapitation, rats were exposed to ether vapors as described. Blood samples were obtained for serum prolactin and progesterone determination by RIA. Prolactin and progesterone determinations Serum prolactin concentration was measured by doubleantibody radioimmunoassay [8] with materials supplied by Dr. A. F. Parlow (National Hormone and Pituitary Program, USA). Prolactin was radioiodinated using the chloramine T method [38] and purified by passage through Sephadex G-75 and polyacrylamide agarose (ACA 54; LKB, Bromma, Sweden) columns. Assay sensitivity was 1 ng/ml serum, and inter- and intra-assay coefficients of variation were
